JPH083063A - Hemoglobin-containing liposome - Google Patents

Abstract

PURPOSE:To produce a hemoglobin-containing liposome excellent in affinity to oxygen and oxidation inhibitory effect and useful as an artificial red blood cell, etc., by admixing, e.g. an enzymatic reaction substrate producing diphosphoglyceric acid with a specified concentrated hemoglobin solution and forming a liposome under a specified condition. CONSTITUTION:This hemoglobin-containing liposome is produced by hemolyzing ashed red blood cells, subsequently removing the membrane components of the red blood cells, adding an enzymatic reaction substrate (preferably 0.1 to 100mM concentration) producing 2,3-diphosphoglyceric acid, further preferably admixing adenosine triphosphate and/or a substrate for an enzymatic reaction producing adenosine triphosphate and nicotinamide adenine dinucleotide of reuduced type, etc., with the concentrated hemoglobin solution of 30 to 60 (W/V)% and forming a liposome therefrom under a mild condition under which enzymes in the hemoglobin solution are not deactivated.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a hemoglobin-containing liposome to be administered in vivo. More specifically, the present invention relates to a hemoglobin-containing liposome having a significantly improved oxygen affinity of hemoglobin when administered in vivo.

[0002]

2. Description of the Related Art As a drug that can substitute for blood, a plasma expander such as a solution of a gelatin decomposition product, a dextran solution or a hydroxyethyl starch solution is used. However, these plasma substitutes are formulations developed for the purpose of supplementing the plasma volume in blood vessels and maintaining hemodynamics in the event of an emergency during massive bleeding, and cannot replace the oxygen carrying capacity of natural red blood cells. In recent years, unlike such a plasma expander, research and development of an artificial oxygen carrier has been promoted as a blood substitute that retains an oxygen carrying capacity similar to natural red blood cells. At the initial development stage, fluorocarbon emulsion (Perfl
uorochemical: PFC [common name: Fluor
The chemical synthesis product utilizing the oxygen-dissolving ability (20 times that of water) was investigated, but there is a concern that the oxygen transport capacity will be insufficient and that bioaccumulation will occur in the body.

On the other hand, attempts have been made to utilize natural erythrocyte-derived hemoglobin as an oxygen carrier, but the half-life of free hemoglobin in vivo is extremely short, oxygen transport to the periphery is low, and there are problems such as nephrotoxicity. It has been pointed out that practical application is difficult. Recently, modified hemoglobin (stabilized hemoglobin, which improves the problems of such free hemoglobin solution,
The focus is on the development of polymerized hemoglobin, etc.) and liposomal hemoglobin.

[0004] As an example thereof, a liposome produced by a thin film method as a liposome containing hemoglobin is Mill.
er et al. (US Pat. No. 4,133,
874). According to this method, a hemoglobin-containing liposome is prepared by dissolving a liposome-forming lipid in an appropriate organic solvent such as chloroform and distilling the solvent off from the resulting solution to form a lipid thin film, which is then added with a hemoglobin solution. Then, it is obtained by ultrasonic treatment.

However, artificial red blood cells made from these hemoglobins are present in natural red blood cells, and by binding to hemoglobin, the oxygen affinity of hemoglobin is adjusted to effectively transport oxygen from the lungs to peripheral tissues. It lacks 2,3-diphosphoglyceric acid, which is a functioning substance (allosteric effector), so it has a very high affinity for oxygen and there is a problem that oxygen is not sufficiently transported in the living body. . For this reason, artificial blood that utilizes free hemoglobin is devised to reduce its affinity by chemically bonding pyridoxal phosphate to hemoglobin or by crosslinking in the molecule.

However, since these modified hemoglobins have different molecular structures from natural hemoglobin, it is considered that there are problems in toxicity and metabolism. In addition, with regard to liposome-containing hemoglobin, attempts have been made to add 2,3-DPG to hemoglobin and inosit hexaphosphate (hereinafter referred to as IHP) known as an allosteric effector of birds. But 2,3-
DPG is very unstable, and IHP has no metabolic pathway in mammals, and it is very strongly associated with divalent ions.

[0007]

The present invention is intended to solve the above problems in view of the above problems. That is, the present invention provides a hemoglobin-containing liposome which has improved oxygen affinity of hemoglobin, and particularly improved hemoglobin's oxygen carrying ability when administered to the body.

[0008]

The above-mentioned problems can be solved by the hemoglobin-containing liposome of the present invention having the following constitution.

(1) A hemoglobin-containing liposome obtained by incorporating a hemoglobin solution having an enzymatic activity into a liposome composed of lipid, wherein 2,3-diphosphoglyceric acid (hereinafter, 2,3-DP) is contained in the hemoglobin solution.
A hemoglobin-containing liposome, characterized in that it contains an enzyme reaction substrate that produces G).

Here, a hemoglobin-containing liposome in which a hemoglobin solution in which the concentration of the enzyme reaction substrate for producing 2,3-DPG is 0.05 mM to 200 mM is incorporated is preferable.

(2) A hemoglobin-containing liposome obtained by incorporating a hemoglobin solution having an enzymatic activity into a liposome composed of a lipid, wherein an enzyme reaction substrate producing 2,3-diphosphoglyceric acid in the hemoglobin solution and adenosine triglyceride Phosphate and / or adenosine triphosphate (hereinafter referred to as ATP)
The hemoglobin-containing liposome according to (1) above, which contains an enzyme reaction substrate that produces

Here, a hemoglobin-containing liposome in which a hemoglobin solution having an enzyme reaction substrate concentration of 0.05 mM to 200 mM is incorporated is preferable.

(3) The above-mentioned (1) and the above-mentioned, wherein the enzyme reaction substrate for producing the 2,3-DPG is an anaerobic glycolytic intermediate or a derivative thereof, or an organic phosphate compound or a derivative thereof. The hemoglobin-containing liposome described in (2).

(4) The anaerobic glycolytic intermediate or derivative thereof is glucose, glucose-6-phosphate, glucose-1,6-diphosphate, fructose-6-phosphate,
UDP-glucose, L-gluconate, xylitol, sorbitol, α-glycerol, gluceraldehyde-3-phosphate, phosphoenolpyruvate, pyruvate, lactic acid, and the organic phosphate compound or its derivative is ATP, adenosine diphosphate. (ADP), adenosine monophosphate (AMP), adenine, inosine, adenosine, the hemoglobin-containing liposome according to (1) and (2).

It is preferable that the concentration of phosphoenolpyruvate added is 1 to 50 mM because the amount of 2,3-DPG produced can be increased and the oxygen transport efficiency can be further improved.

(5) The hemoglobin solution further contains an organic phosphoric acid compound and an inorganic phosphoric acid compound (1)
The hemoglobin-containing liposome according to (4).

Here, the hemoglobin-containing liposome in which the content (weight molar ratio) of the organic phosphate compound is 0.05 to 4 mol per 1 mol of hemoglobin is preferable.

Here, the concentration of the inorganic phosphate compound is 0.1 m.
Hemoglobin-containing liposomes with M-100 mM are preferred. Furthermore, the hemoglobin-containing liposome in which the organophosphate compound is inosit hexaphosphate is preferable.

(6) In the hemoglobin solution, reduced nicotinamide adenine dinucleotide (hereinafter referred to as NA
DH), oxidized nicotinamide adenine dinucleotide (hereinafter referred to as NAD), reduced nicotinamide adenine dinucleotide phosphate (hereinafter referred to as NADPH), oxidized nicotinamide adenine dinucleotide phosphate (hereinafter referred to as NADP) The hemoglobin-containing liposome according to the above (1) to (5), which contains at least one selected from the group consisting of

(7) After washing the washed erythrocytes, the erythrocyte membrane components are removed, and an enzyme reaction substrate for producing 2,3-diphosphoglyceric acid is added to a concentrated hemoglobin solution of 30 to 60% (w / v). The method for producing a hemoglobin-containing liposome according to any one of the above (1) to (6), characterized in that the liposome is formed into a liposome under mild conditions in which the enzyme is not inactivated in the hemoglobin solution.

Here, the mild condition in which the enzyme is not inactivated in the hemoglobin solution is 50 to 1800 kg / at 40 ° C. or lower.
A preferred method for producing hemoglobin-containing liposomes is to eject from the slit one to several times at a pressure of cm ² .

In order to solve the problem of oxygen affinity of hemoglobin-containing liposomes, the present invention uses 2,3-DP
An enzyme reaction substrate that produces G is added to improve the oxygen carrying capacity of the hemoglobin-containing liposome in vivo.

Further, the present invention has been made to solve the problem of oxygen affinity of hemoglobin-containing liposomes by using a few
-DPG, and ATP and / or an enzyme reaction substrate that produces ATP are added to improve the oxygen-carrying ability of the hemoglobin-containing liposome in vivo.

The enzyme reaction substrate that produces 2,3-DPG and / or ATP used in the present invention is not particularly limited, and one or a combination of enzymes existing in the living body and an enzyme formed by a combination of one or more of the substrates. The reaction may be 2,3-DPG or ATP, but a glycolytic intermediate or a derivative thereof and an organic phosphate compound or a derivative thereof are preferable.

Among them, glucose, glucose-6-phosphate, glucose-1,6-diphosphate, fructose-6-phosphate, which are anaerobic glycolytic sugar intermediates,
UDP-glucose, L-gluconate, xylitol, sorbitol, α-glycerol, gluceraldehyde-3-phosphate, phosphoenolpyruvate, pyruvate, lactic acid and the like can be used.

ATP, adenosine diphosphate (ADP), adenosine monophosphate (AMP) can be used as the organic phosphate compound, and adenine, inosine, adenosine, etc. can be used as the derivative thereof. That is, any enzyme can be used as long as it produces 2,3-DPG and / or ATP in the enzymatic reaction of one or a combination of enzymes existing in the living body.

In the present invention, 2,3-DPG and / or
Alternatively, the concentration of the enzyme reaction substrate that produces ATP is 0.01 mM to 200 mM, preferably 0.1 mM to 10 mM.
It is 0 mM. If it is less than 0.01 mM, the oxygen affinity adjusting effect is small, and if it is more than 200 mM, the liposome-forming efficiency is lowered, which is not preferable.

The lipid raw material of the liposome used in the present invention is not particularly limited as long as it can form liposomes, and natural and synthetic ones can be used, examples of which include lecithin, phosphatidylethanolamine, phosphatidic acid, Examples include phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, sphingomyelin and the like, which are hydrogenated according to a conventional method, and these can be used in combination.

The hemoglobin used in the present invention is hemoglobin hemolyzed by a conventional method to remove the membrane, and then concentrated to 30% (w / v) or more by ultrafiltration using a membrane having a molecular weight cut off of 10,000. used. Hemoglobin is incorporated in the form of an aqueous solution, and its concentration is preferably 30 to 60% (w / v), and particularly preferably 40 to 50% (w / v). The reason for setting this concentration is that the amount of oxygen transported to peripheral tissues increases depending on the hemoglobin concentration in the liposome, so that it is preferably the highest concentration within the range where liposome formation is possible.

The erythrocytes used in the present invention are preferably of human origin when administered to humans, and fresh ones are preferable, but those that have been stored at 4 ° C. or lower for 50 days or more after blood collection can be sufficiently used.

In the present invention, not only the above-mentioned enzyme reaction substrate but also NADH, NAD, NADPH, N
A coenzyme such as ADP may be added to the hemoglobin solution.
The concentration is preferably 0.01 mM to 10 mM, respectively.

In the present invention, a charge-giving substance such as sterols and fatty acids can be added as a constituent of the liposome membrane, if desired, to strengthen the membrane structure and adjust the elimination time in the body. In addition, tocopherol homologue, that is, vitamin E may be added to the liposome in order to prevent the oxidation of hemoglobin.

The hemoglobin-containing liposome of the present invention comprises:
After hemolyzing the washed red blood cells, the red blood cell membrane components are removed, and 30 to 6
It is obtained by concentrating it to 0% (w / v), adding one or more enzyme substrates to this concentrated hemoglobin solution, mixing them, and forming liposomes under mild conditions in which the enzyme in the hemoglobin solution is not deactivated. Can be done. The mild condition without enzyme deactivation is preferably high pressure discharge treatment, and discharge from the slit one to several times at a pressure of 50 to 1800 kg / cm ² . Further, the processing temperature is maintained at 40 ° C. or lower. As a specific example, a high pressure discharge emulsifying machine is equipped with a Microfluidizer 110Y [Micorofluidizer 110Y].
(Microfluidizer Co. [maidrofluidics Co.]) 480 to 1450 kg / cm ² , and Pearl cell disrupter (Pearl [Parr Co.]) 80
˜200 kg / cm ² , both are 40 ° C. or lower, preferably under ice cooling ˜4 ° C.

80 to 100 μg / ml in natural red blood cells
There is 2,3-DPG in blood, and its amount is kept almost constant. 2,3-DPG strongly binds to hemoglobin, and the oxygen affinity of hemoglobin is p50 (oxygen partial pressure at which the oxygen saturation of hemoglobin is 50%) = 27 to 30 m
In order to maintain mHg, oxygen is efficiently transported by red blood cells from the lungs to peripheral cells. In addition, since 2,3-DPG binds more strongly to deoxygenated hemoglobin, an S-shaped oxygen dissociation curve (allosteric effect), which is a characteristic of natural erythrocytes, occurs and the oxygen partial pressure of venous oxygen is increased. It contributes to the homeostasis of the living body by the sensitive reaction of transport efficiency. However, this 2,3-DPG is not detected in hemoglobin from which blood has been removed or blood cell membrane has been removed.
Is rarely present and its oxygen affinity is so high that oxygen cannot be effectively transported.

However, as a result of the earnest studies by the present inventor, hemoglobin was liposome-formed with an enzyme substrate in a hemoglobin-containing liposome under a mild condition so that the glycolytic enzyme present in natural erythrocytes was not inactivated. 2,3-
It was able to produce DPG and succeeded in making its oxygen carrying capacity equal to or higher than that of natural red blood cells.

The mechanism is considered as follows.
In other words, if the liposomes are formed under mild conditions as described above, the enzyme remaining in the hemoglobin solution will not be inactivated, but the process of removing membrane components from natural erythrocytes and extracting hemoglobin, or the process of purification and concentration. Also, substances necessary for the enzymatic reaction such as enzyme substrates and coenzymes having a molecular weight of 10,000 or less are lost. Therefore, if it is made into liposome as it is, the glycolytic system does not move and the intermediate of the glycolytic system, 2,3-DPG, is not produced. Therefore, glucose in the hemoglobin solution,
When adenine, inosine, NAD, phosphoenolpyruvate, inorganic phosphate, etc. are added to form liposomes, first, ATP is produced from adenine, inosine, inorganic phosphate, and the glycolytic system of glucose starts using these ATP and NAD as coenzymes. As a result, 2,3-DPG is produced in the liposome, whereby the oxygen affinity of the hemoglobin-containing liposome can be adjusted. Further, when the concentration of phosphoenolpyruvate, which is a metabolic intermediate, is increased, a reverse reaction occurs in the glycolysis system, the amount of 2,3-DPG produced increases, and the oxygen transport efficiency can be increased. In addition, like natural red blood cells, NA
Since a reduction reaction from D to NADH also occurs, methemoglobin generated in the liposome is also reduced by this NADH-cytochrome b5 reductase, and it is presumed that it also contributes to the suppression of the temporal oxidation of hemoglobin.

The present inventor effectively utilizes the glycolytic enzyme activity derived from natural erythrocytes, which has been conventionally regarded as an impurity in hemoglobin solutions from which red blood cell membrane components have been removed, and replenishes necessary substrates and coenzymes. As a result, glycolysis-derived 2,3-DPG similar to natural erythrocytes was generated in the hemoglobin-containing liposome to obtain a hemoglobin-containing liposome with its oxygen affinity adjusted appropriately.

[0038]

EXAMPLES Hereinafter, specific examples will be described based on examples of the present invention. In addition, unless otherwise specified, each step of the preparation was kept in a refrigerated state (4 ° C.) and carried out in an aseptic environment. In addition, reagents and instruments are sterilized, and aseptic ultrapure water (15 megΩ ° C
-Cm at 25 ° C or higher (pyrogen-free)) was used for the preparation.

Example 1 Preparation of Erythrocyte Membrane Removal Hemoglobin Solution (SFH) Concentrated red blood cell preparation 15L. Was washed with physiological saline using a continuous centrifuge to obtain washed red blood cells from which plasma components such as mixed platelets and white blood cells were removed. This washed red blood cell 5L. Against pure water 10 L. Was added to cause hemolysis. Pore size 0.45μ
red blood cell membrane-removing hemoglobin solution (SFH solution) with a hemoglobin concentration of 8% (w / v), using a plasma separator with a molecular weight cut-off of 300,000 (pore size: 5 nm) and a filter having a hemoglobin concentration of 8%. Was recovered. SFH obtained
The solution was adjusted to pH 7.4 by adding sodium bicarbonate, dialyzed against pure water using a hollow fiber type dialyzer (CL-C8N manufactured by Terumo Corp.), and then concentrated by ultrafiltration. And hemoglobin concentration 50%
(W / v) concentrated SFH solution, 1.8 L. I got

Addition of Reagent to Concentrated SFH Solution To 200 ml of concentrated SFH solution prepared through the above steps, β-NAD (1 mM, 133 mg), D-glucose (100 mM, 3.6 g), adenine (2 mM, 5
4mg), inosine (5mM, 268mg), magnesium chloride hexahydrate (1mM, 40mg), potassium dihydrogenphosphate (9mM, 247mg), disodium hydrogenphosphate (11mM, 310mg) to homogenize As mixed.

A uniform mixed powder [HSPE: Chol] of purified soybean-derived phosphatidylcholine (HSPC), cholesterol (Chol), myristic acid (MA), and tocopherol (Toc) of the concentrated SFH solution having a liposome hydrogenation rate of 90% or more. : MA: Toc = 7: 7: 2: 0.28] 45
45 ml of pure water was added to g, and the mixture was heated to 60-70 ° C. and swollen. SFH prepared in was added to the swollen lipid, and the mixture was stirred and mixed (15 seconds). This lipid-concentrated SFH mixture was added to a microfluidizer (Microfluidics co., Microfluid
idizer 110Y), under ice cooling, 12000psi (about 8
Liposomes were formed at a pressure of 44 kg / cm ² ).

Purification of Hemoglobin-Containing Liposomes An equal amount of dextran-added physiological saline (3% (w / v)) was added to the treatment solution of the above example, and the mixture was centrifuged (10
000 rpm (13000 g) × 30 minutes, at 4 ° C.). The liposome containing hemoglobin efficiently was collected as a precipitate by this centrifugation. The supernatant containing free hemoglobin that was not formed into liposomes and the raw material lipid was removed by decantation or suction. After the above washing operation was repeated until hemoglobin in the supernatant was not visually recognized, a dura of 0.45 μm was added. Pore membrane filter (Millipore Ltd., Minit
an system) to remove coarse particles mixed in the suspension. The filtrate was ultrafiltered with a hollow fiber type dialyzer (CL-C8N manufactured by Terumo Corp.) and concentrated to obtain 600 ml of a purified hemoglobin-containing liposome suspension having a hemoglobin concentration of 5% (w / v).

Example 2 For 200 ml of the concentrated SFH solution prepared in Example 1-β-NAD (1 mM, 133 mg), D-glucose (100 mM, 3.6 g), adenine (2 mM, 54)
mg), inosine (5 mM, 268 mg), phosphoenolpyruvate Na (5 mM, 234 mg), magnesium chloride hexahydrate (1 mM, 40 mg), potassium dihydrogen phosphate (9 mM, 247 mg), disodium hydrogen phosphate. (11 mM, 310 mg) was added and mixed so as to be uniform, and liposome formation, purification and concentration were carried out in the same manner as in Example 1 to obtain 600 ml of a purified hemoglobin-containing liposome suspension having a hemoglobin concentration of 5% (w / v). Obtained.

Example 3 To 200 ml of the concentrated SFH solution prepared in Example 1-β-NAD (1 mM, 133 mg), D-glucose (100 mM, 3.6 g), adenine (2 mM, 54)
mg), inosine (5 mM, 268 mg), phosphoenolpyruvate Na (10 mM, 468 mg), magnesium chloride hexahydrate (1 mM, 40 mg), phosphoric acid 2
Potassium hydrogen (9 mM, 247 mg) and disodium hydrogen phosphate (11 mM, 310 mg) were added and mixed so as to be uniform, and liposome formation, purification and concentration were carried out in the same manner as in Example 1, and hemoglobin concentration was 5% (w. 600 ml of the purified hemoglobin-containing liposome suspension of / v) was obtained.

Example 4 For 200 ml of the concentrated SFH solution prepared in Example 1-β-NAD (1 mM, 133 mg), D-glucose (100 mM, 3.6 g), adenine (2 mM, 54)
mg), inosine (5 mM, 268 mg), phosphoenolpyruvate Na (20 mM, 936 mg), magnesium chloride hexahydrate (1 mM, 40 mg), phosphoric acid 2
Potassium hydrogen (9 mM, 247 mg) and disodium hydrogen phosphate (11 mM, 310 mg) were added and mixed so as to be uniform, and liposome formation, purification and concentration were carried out in the same manner as in Example 1, and hemoglobin concentration was 5% (w. 600 ml of the purified hemoglobin-containing liposome suspension of / v) was obtained.

Comparative Example Concentrated S prepared according to the example
600 m of liposome-containing liposome suspension containing control purified hemoglobin having a hemoglobin concentration of 5% (w / v) by adding liposomes to FH and purifying by the same method.
1 was obtained.

(Test Example 1) Measurement of ATP amount in liposomes 37 ml of 10 ml of the above hemoglobin-containing liposome suspension was used.
Incubate at ℃, sample 0.5 ml over time, and add 10% (w / v) Triton X100 to it.
-HEPES buffer solution (0.5 M, pH 7.4) (2 ml) was added and stirred, and then Freon (2 ml) was further added and stirred for about 1 minute. Centrifuge this solution at 3000 rpm for 15 minutes,
1.8 ml of the supernatant was deproteinized with an ultrafiltration membrane having a molecular weight cut off of 5000. The deproteinized solution was quantified for the amount of ATP by high performance liquid chromatography using an ODS column (Senshu Kagaku, ODS-1301-N). The results are shown in Fig. 1.

(Test Example 2) Measurement of 2,3-DPG amount in liposomes 10 ml of the above hemoglobin-containing liposome suspension was added to 37
Incubate at ℃, sample 0.5 ml over time, and add 10% (w / v) Triton X100 to it.
-HEPES buffer solution (0.5 M, pH 7.4) (2 ml) was added and stirred, and then Freon (2 ml) was further added and stirred for about 1 minute. Centrifuge this solution at 3000 rpm for 15 minutes,
1.8 ml of the supernatant was deproteinized with an ultrafiltration membrane having a molecular weight cut off of 5000. The results of measuring the concentration of 2,3-DPG in the deproteinized solution by the 2,3-DPG test "BMY" (Boehringer Mannheim Co., Ltd.) are shown in FIG.

(Test Example 3) The oxygen affinity (p50) of the hemoglobin-containing liposome was measured.
Measurement The above hemoglobin-containing liposome was adjusted to pH 7.4 (at 37%) so that the hemoglobin concentration was 0.05% (w / v).
C., pCO ₂ = 40 mmHg) in a phosphate buffer, and the oxygen partial pressure is 300 mmHg to 0 mmHg (pCO 2
₂ = 40 mmHg) and measure the absorption spectrum at that time to measure oxygen saturation and oxygen affinity (oxygen saturation 50
% Oxygen partial pressure: p50) and oxygen delivery efficiency (lung oxygen partial pressure 100 mmHg, peripheral tissue oxygen partial pressure 40
The ratio of hemoglobin carrying oxygen (OTE) was calculated in mmHg. The results are shown in Table 1 and FIG.

[0050]

[Table 1]

(Test Example 4) Measurement of time-dependent oxidation of hemoglobin 1 ml of the above-mentioned hemoglobin-containing liposome suspension having a hemoglobin concentration of 5% (w / v) was incubated at 37 ° C. and 24 hours later, the oxidation rate was calculated by the following method. did.

The hemoglobin-containing liposome was strong against osmotic pressure difference, and this characteristic was utilized to separate it from natural red blood cells. That is, 8 to 10 volumes of pure water was added to heparin-added whole blood, and the mixture was stirred to hemolyze mouse-derived red blood cells.
High-speed centrifugation was performed at rpm for 30 minutes (4 ° C.) to collect the hemoglobin-containing liposome as a precipitate. This operation was repeated 3 times to completely remove blood components by washing. 10% (w / v) Triton X100-HEP in the obtained sample
After adding 2 ml of ES buffer solution (0.5 M, pH 7.4) and stirring, 2 ml of Freon was further added and stirred for about 1 minute.
This solution was centrifuged at 3000 rpm for 15 minutes and the supernatant 1.
HEPES buffer solution (0.5 M, pH 7.4) for 8 ml
1 ml of the solution prepared in the visible region (700 nm ~
The absorption rate at 460 nm) was measured to calculate the oxidation rate. FIG. 4 shows the results.

[0053]

INDUSTRIAL APPLICABILITY The hemoglobin-containing liposome of the present invention has 2,3-DPG generated in the liposome and has excellent oxygen affinity and oxidation inhibiting ability of the hemoglobin-containing liposome. Furthermore, by increasing the amount of ATP in the liposome by adding ATP or an enzyme reaction substrate that produces ATP, the glycolysis system of glucose starts to move due to the consumption of this ATP, resulting in 2,3-DP.
More G is produced in the liposome.

Therefore, the hemoglobin-containing liposome of the present invention has excellent oxygen affinity and ability to suppress methemoglobin formation, and is effective as an artificial red blood cell.

[Brief description of drawings]

FIG. 1 shows the measurement results of the amount of ATP in liposomes.

FIG. 2 shows the measurement results of the amount of 2,3-DPG in the liposome.

FIG. 3 shows the measurement results of oxygen affinity (p50) in liposomes.

FIG. 4 shows the measurement results of time-dependent oxidation of hemoglobin in liposomes.

Claims (4)

[Claims] 1. A hemoglobin-containing liposome in which a hemoglobin solution having an enzymatic activity is incorporated into a liposome composed of lipid, wherein the hemoglobin solution comprises:
A hemoglobin-containing liposome, which comprises an enzyme reaction substrate that produces 2,3-diphosphoglyceric acid. 2. A hemoglobin-containing liposome obtained by incorporating a hemoglobin solution having an enzymatic activity into a liposome composed of a lipid, wherein the hemoglobin solution comprises:
An enzyme reaction substrate that produces 2,3-diphosphoglyceric acid,
The hemoglobin-containing liposome according to claim 1, which further comprises an adenosine triphosphate and / or an enzyme reaction substrate that produces adenosine triphosphate. 3. A reduced-type nicotinamide adenine dinucleotide, an oxidized-type nicotinamide adenine dinucleotide, and a reduced-type nicotinamide adenine dinucleotide are further added to a hemoglobin solution to which an enzyme reaction substrate that produces 2,3-diphosphoglyceric acid is added. The hemoglobin-containing liposome according to claim 1 or 2, which contains at least one member selected from the group consisting of phosphoric acid and oxidized nicotinamide adenine dinucleotide phosphate. 4. After hemolyzing washed red blood cells, the red blood cell membrane component is removed, and a concentrated hemoglobin solution of 30 to 60% (w / v) is added,
4. An enzyme reaction substrate or the like that produces 2,3-diphosphoglyceric acid is added and mixed to form a liposome under mild conditions in which there is no enzyme inactivation in the hemoglobin solution. A method for producing a hemoglobin-containing liposome.